Mre11, Nbs1 and Rad50 form a conserved protein complex that is required for the maintenance of genome stability. In humans, Nbs1 and Mre11 are linked to the Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD), respectively, and the affected patients are predisposed to cancer. The Mre11/Rad50/Nbs1 complex (MRN) plays a critical role in DNA double stranded break (DSB) repair and cell cycle checkpoint control, but the detailed mechanisms of how these functions are regulated during the cell cycle and in response to DNA damage are not clear. The entire genome needs to be faithfully replicated in S-phase, wherein genotoxic insults most easily occur during the period of active DNA metabolism. Thus, preserving genome integrity in S-phase is most demanding. Our proposed studies will focus on the investigation to understand the mechanisms underlying the critical roles of the Mre11/Rad50/Nbs1 complex (MRN) in preserving genome integrity, especially in mediating S-phase-associated damage responses. First, we will identify DNA damage-induced Mre11 phosphorylation by mass spectrometry analysis and investigate the biological significance of these phosphorylation events in intra-S- phase checkpoint control and DNA DSB repair. Second, we will determine the role of MRN in DSB repair and replication restart at collapsed forks. Since replication forks can stall and collapse when encountering replication obstacles, the function of MRN in repairing DSB at collapsed replication forks is critical for maintaining genome integrity in S-phase. Third, we will investigate the molecular basis underlying the role of MRN in the S-phase associated damage response. We will study the association of MRN with replication forks and understand the role of this interaction in checkpoint activation and damage repair in S-phase related events. These studies will provide significant insights into the molecular mechanisms underlying the critical function of MRN in the maintenance of genome stability and will shed light on how malfunction of this complex could lead to human diseases associated with cancer. PUBLIC HEALTH RELEVANCE: Mutations in Nbs1 and Mre11 genes lead to human diseases, Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD), respectively, and the affected patients are predisposed to cancer. Understanding the role of the Mre11/Rad50/Nbs1 complex in DNA damage response and DNA damage repair will shed light on the cellular mechanisms that prevent genome instability and cancer. These studies will ultimately help develop therapeutic interventions for human diseases associated with genome instability and cancer.